Information gained during in vitro cell culture experiments can profitably be used in the design or selection of materials for use in specific biomaterials such as vascular prostheses. The attachment and growth of endothelial cells and other anchorage-dependent animal cells during in vitro cell culture requires both a suitable substratum for cell attachment and a culture medium that contains either serum, or certain purified serum proteins.
Concerning the chemical nature of the substratum, cells such as endothelial cells do not attach and grow well on hydrophobic surfaces such as nonwettable polystyrene (bacteriological plastic) or on PTFE which is commonly used in vascular prosthetic grafts. On the other hand, cells including endothelial cells also fail to adhere to many hydrophilic polymers, such as the hydrogel poly-2-hydroxyethylmethacrylate, polyHEMA. Cells do attach and grow on polymers where the surface is composed of microdomains containing both hydrophobic and hydrophilic regions. The use of polymers with a microdomain structure of this nature is now the state of the art in the biomedical material area (e.g. the polyurethanes sold under the Registered Trade Marks BIOMER and MITRATHANE). The perfluorosulphonate ionomer which is known by the Registered Trade Mark NAFION has recently been shown to be suitable for endothelial cell attachment and growth (International patent application PCT/AU88/00368; McAuslan et al., (1988) J. Biomed. Mater. Res., 22,963-976; Norris et al., (1988) Clinical Materials, 3,153-162; and may also fit this generalisation, in that as only 1 in every 8 monomer units is sulphonated, the large segments of uncharged chains may allow for both hydrophobic and hydrophilic interactions.
The surface that is commonly used for animal cell attachment and growth in vitro is polystyrene, modified by one of a number of techniques to produce a surface that can promote cell attachment (tissue culture polystyrene). This modification of polystyrene has been performed by treatment with sulphuric acid (Kennedy & Axelrod, (1971) Immunology, 20,253-257); with chromic acid or with sulphuric acid and chromic acid (Klemperer & Knox, (1977) Laboratory Practice 26(3), 179-180); or treatment with a corona discharge process (Maroudas, (1973) in "New Techniques in Biophysics and Cell Biology" (R. H. Payne and B. J. Smith, eds) Wiley Interscience, London). These treatments are believed to introduce hydroxyl groups, and the surface concentration of hydroxyl groups must fall within a range for the polystyrene derivative to be suitable for cell attachment (Curtis et. al., (1983) J. Cell Biology 97, 1500-1506; and Curtis et. al. (1986) J. Cell Sci. 86,9-24). Carboxyl groups produced in the reactions appear to play only a small role in the cell adhesion to modified polystyrene (Curtis et al., 1986). Very few sulphonate groups are introduced into the surface (Curtis et al. 1983).
Somewhat different results as to the surface groups required for cell attachment were obtained in a study of cell attachment to polyHEMA by McAuslan et al (PCT/AU87/00043 and McAuslan et al., J. Biomed. Mater. Res., 1987). In that study it was shown that hydrolytic etching of polyHEMA with sulphuric acid converted the non-adhesive surface into a surface that is highly adhesive for cells. In that case, the improvement in adhesiveness of the hydroxyl-rich surface of polyHEMA surface for cells appeared to correlate with the partial introduction of carboxyl groups onto the surface.
Another aspect of the mechanism of adhesion of cells to polymeric surfaces is that serum adhesive proteins adsorbed to the surface contribute to the cellular attachment reaction. For tissue culture polystyrene, the serum component fibronectin (Fn) has been shown to support endothelial cell attachment. Recent results from Underwood et al. (Aust. New Zealand Soc. Cell Biol., 1988 Meeting, abstracts 1988) point to the adsorption to the polystyrene surface of a second serum component, vitronectins as being essential to the attachment of endothelial cells. The nature of the surface chemistry can have subtle effects on the conformation of the attached serum components with consequential effects on the biological potency of the adsorbed protein. Grinnel and Feld (1981) J. Biomed. Mater. Res., 15, 363-381 and (1982) J. Biol Chem., 257, 4888-4893; have compared the binding of fibronectin to tissue culture polystyrene and biological potency of the bound fibronectin with the binding to hydrophobic unmodified polystyrene. That study showed that the ability of the fibronectin adsorbed to the tissue culture polystyrene surface to promote cell attachment was markedly greater than that of fibronectin adsorbed to the hydrophobic polystyrene surface. It follows that the suitability of a polymer surface for cell attachment is related to both the surface chemistry and to the ability of the surface to adsorb specific adhesive proteins (whether from the serum or as purified serum components) in an active conformation.
The luminal surface of natural blood vessels has an antithrombogenic character which is believed to be a direct consequence of the ability of the endothelial cells that line the vessel to resist thrombus formation. Synthetic vascular grafts have a markedly more thrombogenic surface and frequently fail because of spontaneous thrombosis. It is believed that if the surface of the graft can be covered with endothelial cells that function physiologically, these cells will form a naturally nonthrombogenic interface between the graft and the blood. The cells that are involved in such a process of endothelialisation could arise through spontaneous coverage from endogenous sources (migrating endothelial cells from cut edges of the adjacent blood vessel, or else from capillaries migrating from the perigraft tissue through the interstices of a porous graft) or by seeding of the graft with endothelium. One aspect of the design of vascular prostheses is therefore to ensure that endothelial cells can attach and grow on the surface, particularly where the graft is for use in small to medium-sized arteries that carry low blood flow. Thus the ability of the polymer surface to support endothelial cell attachment and growth is an important characteristic of the effectiveness of the prosthesis. Surfaces that are suitable for endothelial cell attachment and growth are likely to support ingrowth of other mesenchymal tissues, and so be suitable for general implant applications including the enhancement of wound closure and anchorage of percutaneous implants.
The failure of the hydrophobic surface of PTFE to adequately support cell attachment, including attachment of endothelial cells exposed to the shear forces involved in blood flow, is a limitation to the use of this material for vascular prostheses. If PTFE could be modified to produce a surface that supported enhanced endothelial cell attachment and growth, the modified surface could be expected to be more suitable than unmodified PTFE for the process of in vivo endothelialisation. PTFE that is modified to be superior to unmodified PTFE for the attachment of endothelial cells would certainly be preferable for use in the new approach (Herring, Gardner and Glover, (1978) Surgery, 84, 498-504) of preseeding grafts with endothelial cells prior to implantation.
While the introduction of strongly bonded surface carboxyl groups to normally hydrophobic fluorocarbon polymers by various high energy techniques of grafting is well known (eg Charpiro & Jendrychowska-Bonamour (1980) Polymer Engineering and Science, 20(3), 202-205) we have found that the resultant surface poorly supports endothelial cell growth. It appears that the even distribution of carboxyl groups provided by these grafting methods is not beneficial to cell attachment, when quite low (around 1%) grafting levels or higher levels are used. In contrast to this finding and the lack of cell attachment to unmodified PTFE and other fluorocarbon polymers, it has now been found by the present inventors that certain acidic treatments of PTFE and other fluorocarbon polymers to which polyacrylic acid chains have been grafted produce surfaces having improved cell attachment and growth properties, without adversely affecting the physical properties of the materials.
Accordingly, the present invention is centred on the development of processes for the chemical modification of PTFE and other fluorocarbon polymers, to produce an implantable surface that supports the attachment and growth of animal (including human) tissue cells, such as fibroblasts and other mesenchymally-derived cells, epithelial cells and endothelial cells. Where it is endothelial cells in contact with the fluoropolymer surface, the surface produced by this process would support the attachment and growth of the endothelial cells into a confluent surface. The attached endothelial cells then present at the blood interface an antithrombogenic surface which inhibits undesirable platelet interactions.